Review Reports

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Please see the attached .pdf file. 

Comments for author File: Comments.pdf

Author Response

Summary: We greatly appreciate the time and effort dedicated to reviewing our manuscript. We have carefully reviewed each reviewer's comment to improve the quality, clarity, and format of the review. Below, you will find the detailed responses and corresponding revisions marked in the resubmitted file.

 

Point-by-point response to the Comments and Suggestions adopted by the authors

 

Comment 1: The review is written in a neutral tone and lacks a personal style and perspective. This is not a criticism but a comment on this narrative review.

 

Response 1: We appreciate the reviewer's observation. We have strived to maintain scientific objectivity while ensuring the clarity of our critical analysis of the current state of Antarctic bioremediation.

 

Comment 2: Table 2 is not cited in the text.

 

Response 2: Thank you for pointing this out. We have ensured that Table 2 is explicitly cited in the text (Section 5.1) to support the discussion of the enzyme profile of Antarctic fungi.

 

Comment 3: Line 22: All gene designations should be italicized throughout the text and tables.

 

Response 3: We agree. We have carefully reviewed the entire manuscript and tables to ensure that all gene designations (e.g., alkB, almA, ladA, nahAc, ndoB) are italicized in accordance with standard genetic nomenclature.

 

Comment 4: Line 69: rapidly: How rapidly? Delete?

 

Response 4: We agree that the term was ambiguous. We have amended the sentence to be more precise regarding hydrocarbon migration, removing the vague term "rapidly" to accurately reflect the physicochemical behavior of these compounds in porous soils.

 

Comment 5: Line 94: Check the use of upper-case letters: Aliphatic Fractions, Alkenes, Quorum Sensing, Cytochrome, Polycyclic Aromatic Hydrocarbons, many enzyme names, and many others. These. These are all common, not proper, nouns

 

Response 5: We apologize for this oversight. We have corrected the capitalization throughout the manuscript. Terms such as "aliphatic fractions," "alkenes," "quorum detection," and general enzyme names (e.g., "monooxygenases") are now lowercase, except at the beginning of a sentence or when referring to specific proper nouns.

 

Comment 6: Line 105 and elsewhere: Insert a space.

 

Response 6: We have thoroughly proofread the manuscript and inserted the necessary spaces between words and units where they were missing.

 

Comment 7: Remove italicization Table 1 sp., 171 strains, 389 sp., Table 3 sp. consortium, 409 strains.

 

Response 7: We have corrected the format. The abbreviations "sp." and "spp.", as well as the words "strains" and "consortium," are no longer italicized when following a genus name (e.g., Rhodococcus sp.), adhering to taxonomic conventions.

 

Comment 8: Figure 3B, Table 1, 228, 411 Temperature -- Insert a character space before the degree symbol.

 

Response 8: We have corrected the format of temperature units. A character space has been inserted before the degree symbol (e.g., 4 °C) throughout the text, tables, and figures.

 

Comment 9: PAH and RHD are defined at least twice in the text. Some abbreviations are defined but not used below, e.g., QS, CMC, SDS.

 

Response 9: We have streamlined the abbreviations. We ensured that PAH and RHD are defined only at their first mention. Furthermore, we have removed definitions for abbreviations that were not used subsequently (such as QS, CMC, and SDS) or ensured they are used if essential to the text.

 

Comment 10: Line 214: Use proper subscripts in CO2 and H2O.

 

Response 10: We have corrected the chemical formulas. All instances of CO2, H2O, CH4, and other chemical compounds now feature proper subscripts throughout the manuscript.

 

Comment 11: Include a list of nonstandard abbreviations examples: RHD, RSD, IARC, CABS, TPH.

 

Response 11: We have included a comprehensive Abbreviations section at the end of the manuscript, listing nonstandard abbreviations (e.g., RHD, CABS, TPH) to enhance readability.

 

Comment 12: Italicization: 226 Thiobacillus, 255 Rhodococcus, 312 Penicillium (delete sp. or change to spp.), Table 3 ex situ, 474 genus names.

 

Response 12: We have ensured that all genus names (e.g., Thiobacillus, Rhodococcus, Penicillium) are italicized throughout the text. We also corrected the usage of "sp." vs. "spp." and ensured Latin terms like in situ and ex situ are formatted consistently according to the journal's guidelines.

 

Comment 13: Line 392: Caps for figure, table.

 

Answer 13: We have standardized the use of capital letters. References to specific "Figure" and "Table" numbers in the text are now consistently capitalized (e.g., "Table 1," "Figure 2").

 

Comment 14: Figure captions vary with respect to lower-case and capitalized words. Use one format

Consistently

 

Response 14: We have standardized the formatting of all figure titles to ensure consistent capitalization and style across all figures.

 

Comment 15: The references are cited in the text out of the numerical order.

 

Response 15: We have renumbered the in-text citations to ensure they appear in strict numerical order (e.g., [1], [2], [3]...) as they occur, matching the order in the References section.

 

Comment 16: Reference 5: The article title is in lowercase.

 

Response 16: We have corrected the formatting of Reference 5 (and verified others) to ensure that the article title adheres to the correct capitalization style required by MDPI.

 

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The review considers the problem of hydrocarbon pollutants in Antarctica, a region with unique environmental conditions where rules of conduct are regulated and, nevertheless, an increase in anthropogenic impact is noted.

The authors note the exponential growth in scientific logistics, research stations, and tourism intensified the risk of environmental contamination. Among the most significant pollutants are petroleum-derived hydrocarbons, which remain the most predominant and persistent threat to Antarctic soils. Given the extreme environments, low temperatures dramatically increase hydrocarbon viscosity and reduce volatilization, leading to long-term persistence in the subsurface and potential toxicity to native flora and macrofauna.

The structure of the review is quite logical, data on the landscape of hydrocarbon pollution, their sources, spatial distribution, bioavailability depending on environmental conditions and emerging problems with this destruction and impact on biota in this region are presented. Of particular importance are data on the bioavailability of hydrocarbons undergoing transformation after entering soils and the ability of microorganisms to degrade the resulting more complex hydrocarbons. Very important are data on the bioavailability of hydrocarbons undergoing transformation after entering the soil, and the ability of microorganisms to degrade the more complex hydrocarbons formed. Given the uniqueness of the region under consideration, the authors note that bioremediation of pollutants using indigenous and metabolically universal microorganisms is most preferable.

The sections on bacterial diversity and metabolic versatility of the most active hydrocarbon destructors under low temperature conditions are quite informative. In this regard, data on the features of their enzymatic pathways, catobolic potential and a set of life strategies in different representatives, including fungi and yeast, are important. According to the authors, consortia are the most effective for remediation of contaminated soils in low-temperature conditions. Moreover, for their creation, the most promising is the preliminary conduct of omix studies. In this regard, you can discuss. The use of omix technologies does not always allow assessing the physiology of a particular species or strain, which should also be taken into account when selecting consortium participants

To develop a bioremediation strategy in Antarctica, it is necessary to take into account regional climatic features, the presence of permafrost, the depth of the "active layer," and other abiotic factors. The authors gave examples of the influence of different taxa that are part of consortia and with different metabolic capabilities, their ability to ensure the remediation of hydrocarbons in a region unique in environmental conditions. Very useful information about the use of bioventilation, biostimulation, as well as "autochthonous bioaugmentation" in the context of minimizing physical disruption of the ecosystem. Analysis of the diversity of anaerobic participants in the destruction of hydrocarbons indicates existing gaps in its study, including taxonomic affiliation, the presence of specific enzymes, mechanisms that ensure the degradation of various hydrocarbons under low-temperature conditions and at the limit of energy sources.

In Conclusion. The review contains useful information on the problem of remediation of carbohydrate-contaminated soils in the Antarctic region, based on a discussion of published data. Numerous publications on this problem in the Arctic region are presented and discussed, plans for further research are proposed. The inclusion in the Review of the latest data on molecular adaptation mechanisms, an assessment of the role of "uncultivated" microorganisms identified using metagenomics, significantly expanded the understanding of the existing problem and allowed to discuss further plans in its solution.

The comments are mostly minor and relate to the design of the publication and the cited literature. The text is not edited, has edits that make it difficult to read. The tables also need to be adjusted; it is not clear which column the data belongs to. Not everywhere references to sources include source data and comply with journal rules (for example, 35, 39, 58, 63, 64, and so on).

Overall, the work will be useful for variety specialists of interdisciplinary fields of knowledge.

 

 

Author Response

Summary: We sincerely thank the reviewer for the comprehensive analysis of our manuscript and for highlighting the relevance of our review regarding anthropogenic impact and the persistence of hydrocarbons in Antarctic soils. We deeply value the reviewer's positive assessment of the manuscript's logical structure and the pertinence of the data presented concerning bioavailability and enzymatic pathways.

 

We have carefully addressed the specific comment regarding the limitations of omics technologies and have corrected all errors related to the publication's layout, table formatting, and bibliographic citations.

 

Point-by-Point Response to Comments and Suggestions:

Comment 1 (Scientific Discussion): "According to the authors, consortia are the most effective for remediation of contaminated soils in low-temperature conditions. Moreover, for their creation, the most promising is the preliminary conduct of omix studies. In this regard, you can discuss. The use of omix technologies does not always allow assessing the physiology of a particular species or strain, which should also be taken into account when selecting consortium participants."

Response 1: We deeply appreciate this insightful observation. We fully agree with the reviewer that, while multi-omics approaches (particularly metagenomics) are effective for identifying the genetic potential and functional diversity of a community, they do not always reflect the actual physiological state or competitive fitness of a specific strain under *in situ* stress conditions. The mere presence of a gene does not guarantee its expression, nor does it guarantee the survival of the organism.

To address this, we have refined the analysis in Section 4.6 (Future Perspectives: From Strains to Synthetic Consortia) and Section 8.2 (The "Unculturable Majority"). We explicitly clarify that genomic data must be validated through physiological characterization (where cultivation is possible) or integrated with metatranscriptomics and metabolomics to confirm that metabolic potential effectively translates into enzymatic activity under cryospheric constraints. We highlight that the most robust consortia are those designed by combining the "genetic reservoir" identified through omics with the physiological resilience of culturable "key engineers," such as *Rhodococcus* and *Pseudomonas*.

Comment 2 (Text Editing): "The text is not edited, has edits that make it difficult to read."

Response 2: We apologize for the oversight in the previous version of the manuscript. We have conducted a comprehensive review of the entire text to remove any residual editing marks, correct typographical errors, and improve the fluency and readability of the English to meet the journal's standards.

Comment 3 (Tables): "The tables also need to be adjusted; it is not clear which column the data belongs to. Not everywhere references to sources include source data and comply with journal rules (for example, 35, 39, 58, 63, 64, and so on)."

Response 3: We have reformatted Tables 1, 3, and 4 to ensure proper alignment. The columns "Organism/Strategy," "Target Substrate," "Mechanism," and "Reference" are now clearly delineated to avoid confusion. Specifically, in Table 1, we have ensured that the association between specific genera (e.g., *Pseudomonas*, *Sphingobium*) and their adaptive mechanisms is visually clear.

Comment 4 (References): "References to sources do not consistently include full source details or comply with the journal's guidelines (e.g., 35, 39, 58, 63, 64, etc.)."

Response 4: We thank the reviewer for identifying these inconsistencies. We have meticulously reviewed the References section. We have corrected the incomplete citations (including references 35, 39, 58, 63, and 64) by adding the missing information—such as volume numbers, page ranges, and DOIs—where applicable. All citations have been formatted to strictly adhere to the journal's style guidelines.

 

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This is a well-timed and relevant review that offers a useful overview of recent progress in Antarctic bioremediation. However, I realized that the manuscript has some scientific and structural issues that hinder its clarity, rigor, and overall impact. Accordingly, I believe the manuscript requires significant revision based on the following comments to better support the reader.

1. The authors should reorganize and streamline the review by fixing section numbering and removing duplicate content, such as the repeated anaerobic section. Additionally, they need to incorporate tables and figures into the narrative with clear captions and proper references in the text.
2. The authors should adopt a more defined thematic structure, such as: Part I: Pollution context and challenges; Part II: Microbial mechanisms (bacteria, fungi, consortia); Part III: Engineering and field applications; Part IV: Future directions (omics, climate adaptation).
3. The authors should include a dedicated section on ‘Limitations and research gaps’ addressing discrepancies between lab and field results, the “unculturable majority’ and access methods, ethical and regulatory challenges in Antarctica, and a comparison of conflicting studies, such as the effectiveness of bioaugmentation versus biostimulation.
4. The authors should extend Section 5 to incorporate additional case studies of fungal-bacterial collaborations.
5. I believe the authors should consider discussing how hyphal networks and extracellular enzymes might be utilized in biopile or in situ configurations.
6. Add a subsection titled “Bioremediation in a warming Antarctica” covering how rising temperatures could both improve and challenge biodegradation processes. Discuss the risks of faster contaminant movement and methane release, and explain the roles of methanotrophs and “biofilters” in mitigation efforts.
7. Authors should make sure that all main claims are backed by recent and pertinent references.
8. The authors should utilize subheadings more effectively to help guide readers through complex topics such as “Bioavailability in Frozen Soils” and “Enzymatic Adaptations to Cold.”
9. The authors should incorporate a table that compares in situ and ex situ strategies, highlighting their advantages, disadvantages, and case study results.
10. The authors should include a conceptual diagram that depicts an integrated “climate-smart bioremediation” framework.

Author Response

Summary: We sincerely appreciate the reviewer’s constructive criticism and the time taken to provide such a detailed roadmap for improving our manuscript. We found the suggestions regarding the structural reorganization and the inclusion of "Climate-Smart" concepts particularly valuable. We have fully restructured the review into four thematic parts, added the requested tables and figures, and expanded the sections on limitations and future climate scenarios. We believe these major revisions have significantly enhanced the clarity, rigor, and impact of the work.

Point-by-point response to Comments and Suggestions:

Comment 1: The authors should reorganize and streamline the review by fixing section numbering and removing duplicate content... Additionally, they need to incorporate tables and figures into the narrative... The authors should adopt a more defined thematic structure, such as: Part I... Part II... Part III... Part IV...

Response 1: We agree that the original structure needed refinement. We have completely reorganized the manuscript following the reviewer’s exact thematic suggestion:

Part I: Pollution context and challenges (Section 2).

Part II: Microbial mechanisms (Sections 4 & 5).

Part III: Engineering and field applications (Section 6).

Part IV: Future directions (Sections 8 & 9). We have also fixed the section numbering and integrated 4 Tables and 4 Figures directly into the relevant text sections with clear captions to support the narrative.

Comment 2: The authors should include a dedicated section on ‘Limitations and research gaps’ addressing discrepancies between lab and field results, the “unculturable majority”... and a comparison of conflicting studies...

Response 2: We have added a new dedicated section, Section 8: Limitations and outstanding challenges. This section now explicitly discusses:

8.1. From lab to field: The scaling gap and the influence of abiotic factors on reproducibility.

8.2. The “unculturable majority”: The role of omics in accessing "microbial dark matter".

8.3. Bioaugmentation vs. Biostimulation: A critical analysis of the efficiency paradox and regulatory constraints.

Comment 3: The authors should extend Section 5 to incorporate additional case studies of fungal-bacterial collaborations... discussing how hyphal networks... might be utilized in biopile or in situ configurations.

Response 3: We have significantly expanded Section 5. Specifically, in Section 5.5 (Fungal-Bacteria consortia in hydrocarbon degradation), we now discuss how fungal hyphae act as "highways" (physical conduits) for bacterial dispersal and pollutant transport in air-water interfaces. We also included references to specific co-cultures (e.g., Penicillium with Stenotrophomonas) and the use of carrier-based immobilization (biochar) to stabilize these consortia in field applications.

Comment 4: Add a subsection titled “Bioremediation in a warming Antarctica” covering how rising temperatures could both improve and challenge biodegradation processes... explain the roles of methanotrophs...

Response 4: We have introduced Section 9.1: Bioremediation in a Warming Antarctica (Drafted as Biorremediación en una Antártida en calentamiento). This subsection addresses the dual effect of thawing: while it may increase metabolic rates, it also mobilizes legacy contaminants and releases trapped methane. We have included a discussion on the use of methanotrophs as "biofilters" to mitigate methane emissions from thawing permafrost during remediation efforts.

Comment 5: The authors should utilize subheadings more effectively... such as “Bioavailability in Frozen Soils” and “Enzymatic Adaptations to Cold.”

Response 5: We have refined the subheadings throughout the text to be more descriptive and guide the reader. Examples include Section 2.2: Chemical Recalcitrance and Bioavailability and Section 7.1: Cold-Active Enzymes: Overcoming the Kinetic Barrier.

Comment 6: The authors should incorporate a table that compares in situ and ex situ strategies, highlighting their advantages, disadvantages, and case study results.

Response 6: We have created and inserted Table 3: Comparison of in situ and ex situ bioremediation strategies in Antarctica. This table provides a side-by-side comparison of strategies (e.g., Biopiles vs. Bioventing), listing specific pros/cons and referencing quantitative results from case studies at stations like Carlini, Casey, and Signy.

Comment 7: The authors should include a conceptual diagram that depicts an integrated “climate-smart bioremediation” framework.

Response 7: We have designed a new figure, Figure 4: Climate-Smart Bioremediation Framework in Contaminated Antarctic Soils. This diagram visualizes the integration of renewable energy, methanotrophic biofilters, and real-time genetic monitoring to ensure that remediation efforts do not contribute to positive climate feedback loops.

Comment 8: Authors should make sure that all main claims are backed by recent and pertinent references.

Response 8: We have conducted a thorough review of our bibliography. The manuscript now prioritizes recent literature, with a significant number of references from the 2020–2025 period, ensuring that our discussion on omics, emerging contaminants, and climate change reflects the current state of the art.

 

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Subheading 9.1. Biorremediación en una Antártida en calentamiento

Some of the authors’ comments are in Spanish

The references section is a mess of many different formats.

Comments on the Quality of English Language

The manuscript should be completely written in English. 

Author Response

Some of the authors’ comments are in Spanish. The references section is a mess of many different formats.
Response: The authors appreciate this comment and confirm that the manuscript has been thoroughly revised and is now entirely written in English. Likewise, all references have been formatted according to the guidelines of the Multidisciplinary Digital Publishing Institute (MDPI). 

Section 9.1: Bioremediation in a warming antarctica

• Spanish comments removal: All internal notes and comments in Spanish used during the collaborative drafting phase have been removed. The entire section is now strictly in academic English, ensuring clarity for the international readership.

  Technical refinement: We have clarified the "Chemical Paradox" mentioned in this section, explaining how seasonal thawing mobilizes legacy contaminants, transferring them from frozen subsurface reservoirs to vulnerable coastal ecosystems.

  Methane mitigation: Following the suggestion to avoid generic statements about "warming," we expanded on the role of methanotrophic biofilters as a "climate-smart" strategy to mitigate greenhouse gas emissions during the degradation process in thawing permafrost.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

I appreciate the authors' significant and careful revisions after my initial feedback. The manuscript has markedly improved in its organization, clarity, and scientific strength. Taking these changes into account, I suggest accepting the manuscript as is.

Author Response

Comments: I appreciate the authors' significant and careful revisions after my initial feedback. The manuscript has markedly improved in its organization, clarity, and scientific strength. Taking these changes into account, I suggest accepting the manuscript as is.
Response: The authors would like to thank the reviewers for their comments, suggestions, and valuable contributions to the manuscript during the review process. Additionally, we would like to indicate that the English language has been carefully revised again to improve the clarity and overall readability of the text.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

 

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Microorganisms form Antarctica: a review of their potential in bioremediation of hydrocarbon-contaminated soils” authors J. Naranjo-Moran, M.F. Ratti and M. Vera-Morales. The article fits the scope of the journal “Microorganisms”. The topic of the manuscript relates to Antarctica hydrocarbon-degrading microorganisms (bacteria and fungi) in the context of bioremediation of hydrocarbon-contaminated soil. Having carefully read the manuscript, I recommend not to publish it in the current version. I found several faults, which significantly decrease the quality of this manuscript.

 

 

1. In some parts the text is chaotic. For example there are some repetitions about the biosurfactants in the section “4. Bacteria in the bioremediation of Antractic soils” (the fragment starting from hydrocarbonoclastic bacteria and line 218 other bacteria can produce biosurfactants). Also, the text regarding in situ and ex situ remediation is repeated. I suggest to reorganize the text, the first part should describe the bacterial strains degrading hydrocarbons isolated from Antarctic soils, then focusing on the strategies used to degrade hydrocarbons more effectively (biosurfactants).
2. There is no summary regarding the most promising microorganisms for hydrocarbon bioremediation of contaminated Anatrctic soils. Add discussion of most promising bacterial groups/strains/genera for bioremediation, which should be priority for additional study.
3. There is no single section about the adaptation strategies to thrive in cold soil environments. There is no information about temperature growth of the presented microorganisms (psychrotolerant/psychrophilic?).
4. The Authors focused mainly on bacteria and fungi, including yeasts. However, the information about the yeasts is rather scare and does not summarized the recent finding (for example please find the reference [96] and the hydrocarbon-degrading yeasts (Section 26.5.1). In my opinion, the section regarding hydrocarbon-degrading yeasts should be rewritten and expanded.
5. The Authors discussed about the biosurfactants, omitting adapting strategies to thieve in hydrocarbon-contaminated soil in cold environment.
6. Both anaerobic and anaerobic hydrocarbon degrading processes are observed. The Authors focused mostly on aerobic microorganisms, putting a low impact on anaerobic hydrocarbon-degrading bacteria. It should be mentioned that only aerobic bacteria were discussed or provide the summary of anaerobic hydrocarbon-degrading bacteria.
7. The hydrocarbon metabolism was marginal.

The Authors focused on hydrocarbon biodegradation therefore I suggest to mention that methane is the simplest hydrocarbons, however the degradation pathways of this compound is different than for higher alkanes and it will not be discussed in this review.

 

8. The data provided in Table 1 are organized by place and reference. It would be beneficial to make connections between bacterial strains/capabilities (degraded hydrocarbons)/growth conditions (temperature). So it would be possible to see and discuss the link between bacteria and their degrading capabilities and discuss the direction for additional research and remediation perspective. The same regards to Table 2.
9. I do not understand the data gathered in column “Mechanism” in Table 1. There are information such as biostumulation, biofilm production, transformation of chemical compounds, enzyme production. This data is chaotic and there is no clue according to which the data is organized. Biostimulation is a bioremediation approach, while enzyme production refers to metabolism. What enzymes do the Authors mean in the term “enzyme production”?

 

 

10. English needs editing in some parts of manuscript (i.e. Table 3: bioestimulation, hidrocarbons).
11. Some Figures are misleading (for example Figure 3). The manuscript is related to the hydrocarbon-degrading fungi (microorganisms) not mashrooms, which are on Fig. 3.
12. It would be interesting to provide the hydrocarbon contamination levels in Antarctica, the Authors wrote only: “Although hydrocarbon contamination levels in the Antarctic continent are relatively low compared to global reports [17], their impact is not insignificant.” (line 65-66).
13. Line 175-177: What was the reason to mention about the hydrocarbonoclastic bacteria in the context of hydrocarbon-contaminated soil in Anarctica? In the cited paper [59] there is no information about those bacteria isolated/found in Antarctica. Hydrocarbonclastic bacteria mainly inhabit marine environment.
14. There is no complete review since findings of some authors were not cited in this manuscript:

BIODEGRADATION ABILITY OF FUNGAL STRAINS ISOLATED FROM ANTARCTICA TOWARDS PAH M.G. GERGINOVA, N.M. PENEVA, E.T. KRUMOVA, and ALEXIEVA Z.A.

 

Shukor et al. 2009. Isolation and characterization of a Pseudomonas diesel-degrading

strain from Antarctica

 

About the bioremediation attempts:

Van Dorst et al. 2021. Microbial community analysis of biopiles in Antarctica provides evidence of successful hydrocarbon biodegradation and initial soil ecosystem recovery

https://doi.org/10.1016/j.envpol.2021.117977

 

Camenzuli and Freidman, 2015. On-site and in situ remediation technologies applicable to petroleum hydrocarbon contaminated sites in the Antarctic and Arctic. https://doi.org/10.3402/polar.v34.24492

 

15. Conclusions are rather the summary of the manuscript. It does not provide the information about challenges regarding the bioremediation of hydrocarbon-contaminated Antarctic soils, about the most promising bacteria/fungi potentially applied in hydrocarbon remediation; perspective for future studies.

 

 

Minor remarks:

Table 2. What do the Author mean by “Eleven species” in the columns “Organism” in Table 2.

Line 92: there is a logical error: every crude oil contains hydrocarbons

Line 133-134: Fuel spills is the accumulation of aliphatic and aromatic compounds (it doesn’t lead to such accumulation).

Line 135: “The aromatic hydrocarbons present include styrene, toluene, ethylbenzene, xylene, naphthalene, and polycyclic hydrocarbons” There is some error: naphthalene is the simples PAHs. It should be rewritten.

Line 147: “(…) The degradation potential depends on environmental factors, the abundance and diversity of microorganisms, and the nature and chemical structure of the contaminant compound” Degradation potential of what? Please clarify.

Line 154-155: Lack of reference.

Line 161: “Bacteria and archaea are the organisms most commonly reported to withstand extreme environments [54].” Do archaea take part in hydrocarbon degradation? Please specify in the introduction what is the aim of this review => if the Authors discuss also the archaea (in my opinion not), please specify it. In this context, I suggest to remove all the information which are not informative in the context of the discussed topic.

Line 200-202 and 210-211: Bacteria can aerobically degrade hydrocarbons through lactone formation and aromatization pathways (Colwellia, Roseovarius, Salinicola, and Nocardioides) [61].

Bacteria can aerobically degrade aromatic hydrocarbons via dioxygenase pathways  forming cis-dihydrodiols [87].

Which sentence is correct?

Something is wrong with this sentence: “Bacteria can aerobically degrade hydrocarbons through lactone formation and aromatization pathways” => how is it possible to make more aromatic compound from aromatic hydrocarbons, such as PAHs?) Moreover, the aromatic compound is rather dearomatized during degradation.

Line 211-213: The dihydrodiols cannot produce intermediate compounds (the non-enzymatic compound cannot produce another compound). Catechols cannot metabolize other compounds, only the enzymes can catabolize the compound.

Figure 2. In the context of metabolism, the correct term is pathway (anaerobic/aerobic pathway) not route. Moreover, “the anaerobic routes” are difficult for interpretation and the message is not very clear (what “nitrate, methanogenic, sulfate” mean?).

Figure 3. Three from four compounds presented on Figure 3 are not hydrocarbons, while the caption is “Main hydrocarbon contaminants in Antarctica…”.

Line 332-334. Something is wrong here: how bacteria can show emulsification index? What do the Authors mean by “emulsification index activity”?

Comments on the Quality of English Language

The English should be polished since there are spelling errors in Table 3. 

Author Response

Response to the reviewer 1

Comment 1: In some parts the text is chaotic. For example there are some repetitions about the biosurfactants in the section “4. Bacteria in the bioremediation of Antractic soils” (the fragment starting from hydrocarbonoclastic bacteria and line 218 other bacteria can produce biosurfactants). Also, the text regarding in situ and ex situ remediation is repeated. I suggest to reorganize the text, the first part should describe the bacterial strains degrading hydrocarbons isolated from Antarctic soils, then focusing on the strategies used to degrade hydrocarbons more effectively (biosurfactants).

Answer 1: We greatly appreciate your thorough analysis and valuable feedback on the manuscript's structure. We fully agree that the repetition of topics such as biosurfactant production and in situ and ex situ remediation strategies fragmented the narrative, making the reading less fluid than desired. We have taken your suggestion into account and reorganized the text to establish a more logical and coherent sequence. The manuscript now first presents the bacterial microorganisms identified in Antarctic soils and subsequently focuses on their specific degradation strategies, such as biosurfactant production. We believe this adjustment not only eliminates redundancies but also creates a clearer connection for the reader between the microorganisms and their mechanisms, significantly improving the quality and impact of the review. References have been added on lines 74, 77, 84, 93, and 99, and a paragraph has been added on lines 112 to 117.

Comment 2: There is no summary regarding the most promising microorganisms for hydrocarbon bioremediation of contaminated Anatrctic soils. Add discussion of most promising bacterial groups/strains/genera for bioremediation, which should be priority for additional study.

Answer 2: We appreciate the reviewer's comments. We understand that the absence of a section dedicated to adaptation strategies and a summary of the most promising microorganisms would compromise the clarity and value of the review.

Comment 3: There is no single section about the adaptation strategies to thrive in cold soil environments. There is no information about temperature growth of the presented microorganisms (psychrotolerant/psychrophilic?).

Answer 3: We have made the following corrections to address these important suggestions: Regarding adaptation strategies: A dedicated section detailing the physiological and molecular adaptations of Antarctic microorganisms to the cold environment has been integrated. This text clearly distinguishes between psychrophilic organisms, which thrive in consistently low temperatures, and psychrotolerant organisms, which withstand wider temperature fluctuations. The section now includes concrete examples of survival mechanisms, such as the production of cryoprotective proteins and the modification of membrane composition to maintain cellular fluidity at low temperatures. Regarding the most promising microorganisms: We have added a detailed summary identifying the bacterial and fungal genera with the greatest potential for bioremediation of contaminated soils in Antarctica. The importance of bacterial genera such as Rhodococcus, Pseudomonas, and Sphingomonas is discussed due to their metabolic capacity and their production of biosurfactants and biofilms, which increase the bioavailability of hydrocarbons. Similarly, the fungal genera Aspergillus and Penicillium are highlighted, along with yeasts such as Pichia caribbica and Exophiala macquariensis, due to their proven effectiveness in the degradation of various hydrocarbons.

Comment 4: The Authors focused mainly on bacteria and fungi, including yeasts. However, the information about the yeasts is rather scare and does not summarized the recent finding (for example please find the reference [96] and the hydrocarbon-degrading yeasts (Section 26.5.1). In my opinion, the section regarding hydrocarbon-degrading yeasts should be rewritten and expanded.

Answer 4: We appreciate the comment. We have expanded and rewritten the section on hydrocarbon-degrading yeasts to include more recent findings. The section now details the ability of yeasts such as Pichia caribbica and Exophiala macquariensis to degrade hydrocarbons such as diesel and toluene. In addition, we have incorporated the work of Martorell et al., which evaluated the phenol and n-hexadecane assimilation capacity of 60 Antarctic yeasts, including the genera Metschnikowia, Candida, and Rhodotorula.

Comment 5: The Authors discussed about the biosurfactants, omitting adapting strategies to thieve in hydrocarbon-contaminated soil in cold environment.

Answer 5: Your suggestion to link these two concepts is fundamental to the article. We have restructured the text to ensure that information on biosurfactants is not presented in isolation, but as a key example of the multiple and remarkable adaptations that Antarctic microorganisms have developed to survive and thrive in cold, contaminated soils.

Comment 6: Both anaerobic and anaerobic hydrocarbon degrading processes are observed. The Authors focused mostly on aerobic microorganisms, putting a low impact on anaerobic hydrocarbon-degrading bacteria. It should be mentioned that only aerobic bacteria were discussed or provide the summary of anaerobic hydrocarbon-degrading bacteria.

Answer 6: I have restructured the section on hydrocarbon degradation to address your comments. The manuscript now recognizes that both aerobic and anaerobic processes are observed in hydrocarbon bioremediation. We have added a section summarizing the mechanisms of anaerobic degradation, although it is noted that this is a less studied area than aerobic degradation in cold environments. Information has been included clarifying that anaerobic degradation is coupled to the reduction of other compounds such as nitrate, sulfate, and iron (III). Additionally, it has been mentioned that bacteria from genera such as Geobacter, Pseudomonas, and Thiobacillus are capable of degrading hydrocarbons under anaerobic conditions at low temperatures, down to -5°C. This new information provides a more complete perspective on bioremediation in Antarctica, while still recognizing that most of the available data focus on aerobic microorganisms [1, 1].

Comment 7: The hydrocarbon metabolism was marginal.

The Authors focused on hydrocarbon biodegradation therefore I suggest to mention that methane is the simplest hydrocarbons, however the degradation pathways of this compound is different than for higher alkanes and it will not be discussed in this review.

Answer 7: We have adjusted the manuscript to ensure that hydrocarbon metabolism, especially alkanes, is not a marginal topic. It is now explicitly stated that methane, as the simplest hydrocarbon, has a distinct degradation pathway than more complex alkanes and will therefore not be discussed in this review, as it focuses on the biodegradation processes of more complex hydrocarbons.

Comment 8: The data provided in Table 1 are organized by place and reference. It would be beneficial to make connections between bacterial strains/capabilities (degraded hydrocarbons)/growth conditions (temperature). So it would be possible to see and discuss the link between bacteria and their degrading capabilities and discuss the direction for additional research and remediation perspective. The same regards to Table 2.

Answer 8: We completely restructured Tables 1 and 2 to improve their clarity and scientific relevance. The tables now directly connect the bacterial/fungal strain with the degraded substrates, growth conditions (temperature, for example), and specific degradation mechanisms (such as biosurfactant or enzyme production). Furthermore, we have eliminated the "Mechanism" column as it conflated concepts of bioremediation (biostimulation) with metabolic processes (enzyme production). The new tables focus exclusively on the intrinsic processes of microorganisms, allowing for a more coherent discussion of the link between their degradation capabilities and the environmental conditions in which they operate, better guiding future research.

Comment 9: I do not understand the data gathered in column “Mechanism” in Table 1. There are information such as biostumulation, biofilm production, transformation of chemical compounds, enzyme production. This data is chaotic and there is no clue according to which the data is organized. Biostimulation is a bioremediation approach, while enzyme production refers to metabolism. What enzymes do the Authors mean in the term “enzyme production”?

Answer 9: In view of this aspect, we have considered restructuring the table in accordance with the comment.

Comment 10: English needs editing in some parts of manuscript (i.e. Table 3: bioestimulation, hidrocarbons).

Answer 10: I have reviewed the manuscript and the reviewer's suggestions. I will make the necessary editorial corrections to ensure the accuracy of the language, including the terms "biostimulation" and "hydrocarbons" in Table 3, in accordance with proper English grammar and scientific usage.

Comment 11: Some Figures are misleading (for example Figure 3). The manuscript is related to the hydrocarbon-degrading fungi (microorganisms) not mashrooms, which are on Fig. 3.

Answer 11: We have acknowledged the criticism that Figure 3 was misleading and have corrected it based on your observations. The original figure depicted mushrooms, a type of macroscopic fungus, in a manuscript that focuses on microorganisms such as filamentous fungi and yeasts, which are the true agents of bioremediation. Furthermore, the figure contained errors in the classification of contaminants. Figure 3 has been replaced with an accurate representation of the microorganisms and contaminants discussed in the manuscript, ensuring that it is scientifically relevant and clear to the reader. The new figure shows the correct contaminants, and its caption has been adjusted to remove any incorrect information.

Comment 12: It would be interesting to provide the hydrocarbon contamination levels in Antarctica, the Authors wrote only: “Although hydrocarbon contamination levels in the Antarctic continent are relatively low compared to global reports [17], their impact is not insignificant.” (line 65-66).

Answer 12: We have adjusted the manuscript to ensure that hydrocarbon metabolism, especially alkanes, is not a marginal topic. It is now explicitly clarified that methane, as the simplest hydrocarbon, has a distinct degradation pathway than more complex alkanes and will therefore not be discussed in this review, as the review focuses on more complex hydrocarbon biodegradation processes.

Comment 13: Line 175-177: What was the reason to mention about the hydrocarbonoclastic bacteria in the context of hydrocarbon-contaminated soil in Anarctica? In the cited paper [59] there is no information about those bacteria isolated/found in Antarctica. Hydrocarbonclastic bacteria mainly inhabit marine environment.

Answer 13: We appreciate your insightful comment. We recognize that the use of the term "hydrocarbonoclastic bacteria" in the context of Antarctic soils was imprecise. As you point out, these microorganisms are specialists that predominate in marine environments, such as Alcanivorax and Marinobacter. We have revised the manuscript to clarify this distinction or remove it, thus ensuring greater scientific accuracy in the review.

Comment 14: There is no complete review since findings of some authors were not cited in this manuscript:

BIODEGRADATION ABILITY OF FUNGAL STRAINS ISOLATED FROM ANTARCTICA TOWARDS PAH M.G. GERGINOVA, N.M. PENEVA, E.T. KRUMOVA, and ALEXIEVA Z.A.

Shukor et al. 2009. Isolation and characterization of a Pseudomonas diesel-degrading strain from Antarctica

About the bioremediation attempts:

Van Dorst et al. 2021. Microbial community analysis of biopiles in Antarctica provides evidence of successful hydrocarbon biodegradation and initial soil ecosystem recovery

https://doi.org/10.1016/j.envpol.2021.117977

Camenzuli and Freidman, 2015. On-site and in situ remediation technologies applicable to petroleum hydrocarbon contaminated sites in the Antarctic and Arctic. https://doi.org/10.3402/polar.v34.24492

Answer 14: I acknowledge and accept your criticism regarding the missing references in the manuscript. The omission of these key works compromised the integrity of the review, preventing the document from providing a complete and up-to-date overview of hydrocarbon bioremediation in Antarctica.

To address this deficiency, I have incorporated the suggested references into the relevant sections. Specifically, I have added information on:

Fungal Biodegradation of PAHs: The work of Gerginova et al. has been cited to include findings on the ability of Antarctic fungal strains to degrade polycyclic aromatic hydrocarbons.

Isolation of Bacteria: I have included the study by Shukor et al., which describes the isolation and characterization of a diesel-degrading Pseudomonas strain from Antarctica, strengthening the bacteria section.

 

Bioremediation Strategies: I have incorporated the research of Van Dorst et al. and Camenzuli & Friedman, which provide crucial evidence on the application of techniques such as biopiles and in situ bioremediation in Antarctica.

Answer 15: Conclusions are rather the summary of the manuscript. It does not provide the information about challenges regarding the bioremediation of hydrocarbon-contaminated Antarctic soils, about the most promising bacteria/fungi potentially applied in hydrocarbon remediation; perspective for future studies.

Answer 15: The rewrite of the conclusion has been completed. The conclusion is no longer a simple summary of the manuscript; instead, it highlights the challenges of bioremediation in Antarctic soils, identifies the most promising microorganisms for future study, and provides a clear perspective for research, focusing on cutting-edge technologies such as metagenomics and proteomics. [1, 13, 1]

 

Response to minor comments

I have reviewed and processed all of the detailed comments. Below, I present a concise and timely response to each of the minor corrections made to the manuscript:

Table 2, "Eleven Species": The "Organism" column has been removed, and the table has been restructured to directly link the fungal strain to the degraded substrates, growth conditions, and specific degradation mechanisms. The information is presented in a clearer and more coherent manner.

Line 92: The phrase "all crude oil contains hydrocarbons" has been rewritten to avoid redundancy and improve the accuracy of the text.

Lines 133-134: The wording has been corrected to clarify that "fuel spills lead to the accumulation of aliphatic and aromatic compounds," rather than defining them as the accumulation itself.

Line 135: The text has been revised to reflect that naphthalene is the simplest of the polycyclic aromatic hydrocarbons (PAHs), correcting the original conceptual error.

Line 147: It has been clarified that the phrase refers to the "degradation potential of microorganisms" or "soil," resolving the noted ambiguity.

Lines 154-155: A bibliographic reference has been added to support the claim about the ability of microbial consortia to mineralize up to 98% of hydrocarbons.

 

Line 161: The reference to archaea has been removed from the introduction to maintain the review's focus on bacteria and fungi, the microorganisms discussed in detail.

Lines 200-202 and 210-211: The conceptual error has been corrected. The description of the "aromatization" process has been removed, and the text now focuses on the dioxygenase pathway leading to the formation of cis-dihydrodiols, which is the correct degradation pathway.

Lines 211-213: It has been clarified that dihydrodiols are transformed by dehydrogenase enzymes into key intermediates such as catechol, correcting the inaccuracy that they "cannot produce intermediate compounds."

Figure 2: The term "pathway" has been replaced with "pathway" or "metabolic route," which is the correct scientific terminology. Clarifications have been added to the legend to better explain the roles of nitrate, sulfate, and methanogenic processes.

Figure 3: The figure showing mushrooms has been replaced, as they are not relevant to the topic of the manuscript, and the figure caption has been corrected to accurately depict contaminating hydrocarbons. Lines 332-334: The sentence has been rewritten to accurately reflect that bacteria produce biosurfactants, and it is these biosurfactants that exhibit the "emulsification index."

 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides the first comprehensive synthesis of Antarctic hydrocarbon-degrading microbial resources and their mechanisms of cold adaptation. It offers a theoretical basis and directions for biotechnological applications in polar pollution remediation and holds significant reference value for research in extreme environment microbiology and ecological restoration. The manuscript's topic is novel, and the data are comprehensive. However, the following issues need to be addressed:

The introduction does not clearly define the objectives of the review. Rewrite the introduction to emphasize the review's framework/structure (e.g., "This review focuses on microbial degradation mechanisms, bottlenecks in remediation technologies, and future directions").

Merge sections 2 and 3 to avoid redundancy.

Re-draw Figure 3 to simplify the flow and add a legend;

Conclusions (lines 381-390) only recapitulate the results and do not distill the technological bottlenecks (e.g., limitations of low-temperature enzyme activity) or cross-disciplinary applications (e.g., the potential of biosurfactants for industrialization).

Add paragraphs discussing forward-looking directions such as "Challenges of engineering microbial remediation in the Antarctic" or "Genomics-assisted strain screening".

Author Response

Response to reviewer 2

We appreciate the reviewer's detailed and constructive comments. We have made the following corrections to the manuscript to address the points raised:

The introduction has been rewritten to more clearly define the objectives of the review and its structure.

Sections 2 and 3 have been merged to eliminate redundancies and improve the flow of the text.

Figure 3 has been completely redesigned to be scientifically accurate, and a clear legend has been added that correctly describes the compounds.

The conclusions have been rewritten to go beyond a summary, distilling technological bottlenecks and highlighting the potential for future applications.

New paragraphs have been added to discuss future prospects, including the challenges of bioremediation engineering in Antarctica and the potential of genomic tools for strain selection.